Although the important role of p53 and mutations in the p53 gene in cancer etiology has been established and gene transfer has advanced enormously in the last ten years only a limited number of studies have addressed the transfer of the p53 gene into malignant cultured cells to render them non-tumorigenic after injection into nude mice; even a smaller number of studies have used transfer of the p53 gene in animal models (for example Ko, et al., 1996). A clinical protocol at M. D. Anderson Cancer Center (Houston, Tex.) proposed the transfer of wt p53 gene with and without cisplatin in non-small cell lung cancer patients shown to have mutations in the p53 gene using adenovirus5-CMV-p53 construct (Roth, 1996) and p53 adenovirus driven by the b-actin promoter (Roth, et al., 1996).
Cells from over 85% of human malignancies are associated with mutations in the p53 tumor suppressor gene. The wild-type (normal, non mutated) p53 gene plays a pivotal role in arresting the cell cycle and the proliferation of cells after severe damage to the DNA; the mechanism is thought to involve the upregulation of the p21/WAF-1/Cip-1 gene (see below) whose product can interact with PCNA (proliferating cell nuclear antigen), an accessory molecule to DNA polymerase d causing inhibition in DNA replication but also by inhibition of CDK (cyclin-dependent kinase) by p21; CDK is responsible for phosphorylating RB at a strategic site causing transversion of the cell cycle through the G1/S checkpoint (Boulikas, 1997).
Furthermore, p53 upregulates the death-inducing gene BAX and down-regulates the Bcl-2 gene promoting cells to enter the apoptotic pathway; mutant p53, such as that found in the majority of human tumors, has lost the capacity of transactivating the p21, BAX and other genes; indeed, the most frequent inactivating mutations occur at the DNA-binding domain of p53 at strategic amino acid sites that contact DNA.
Although the cause versus effect of p53 mutations on human malignancies is not clear for many cases, overexpression of an exogenous wild-type (wt) p53 gene followed by suppression of the endogenous mutated gene of p53 that can have an antagonizing effect to wt p53, is an approach found to suppress tumorigenicity of cells in culture and is proposed here as a method for cancer treatment.
Central to this patent is our demonstrated ability to specifically target tumor cells in culture using the luciferase reporter gene, as well therapeutically important genes, in supercoiled plasmids under control of tissue-specific and tumor specific control elements including but not limited to matrix-attached regions (MARs) (Boulikas, et al., in preparation).
MARs are claimed as largely responsible for mediating the effects of p53 on cell cycle arrest both in the sense that the regulatory regions of p53 targets (p21, BAX, PCNA regulatory regions) might be associated with the nuclear matrix as well as by the established property of MARs to be enriched in triplex-, cruciform- and other unusual DNA structures including stretches of cruciforms with insertion deletion mismatches. We claim that wt and mutant (mu) p53 interact differently with MAR DNA.
I. p53 as a Tumor Suppressor Protein
Alterations in the p53 tumor suppressor gene appear to be involved, directly or indirectly, in the majority of human malignancies (Vogelstein, 1990). Both alleles of p53 need to be mutated or altered for transformation. Introduction of a null mutation by homologous recombination in murine embryonic stem cells gave mice which appeared normal but were susceptible to a variety of neoplasms by 6 months of age (Donehower, et al., 1992; Harvey, et al., 1993).
The tumor suppressive activity of p53 seems to involve at least four independent pathways: (i) upregulation of specific genes most important of which appears to be p21; p21 up-regulation inhibits the activity of cyclin-dependent kinases (CDKs) leading to inability to phosphorylate RB and to release E2F from its complex with RB; released E2F upregulates genes whose products are needed for DNA synthesis (reviewed by Boulikas, 1995e). p21 induction also leads to p21 association with proliferating cell nuclear antigen (PCNA) leading to the inactivation of the PCNA function as an auxiliary factor for DNA polymerase d and to arrest in DNA synthesis in S phase; (ii) induction of the death-promoting bax gene and down-regulation of bcl-2 gene as a mechanism which eliminates oncogenic virus-infected and transformed cells and as an important mediator of apoptosis during embryogenesis and in B cell maturation; (iii) direct interaction of p53 with origins or replication preventing firing and initiation of DNA replication; (iv) induction of Gadd45 leading to growth arrest; (v) a role of p53 in DNA repair; p53 is believed to patrol the genome for small insertion deletion mismatches (Lee, et al., 1995) or free ends of DNA able to attract RPA, an accessory to DNA polymerases a and d as well as TFIIH at the damaged sites (both TFIIH and RPA have a demonstrated role in DNA repair) and to induce arrest in the cell cycle or apoptosis after DNA damage (see below); (vi) upregulation of the gene of thrombospondin inhibiting which inhibits neovascularization in solid tumors (Dameron, et al., 1994); and (vii) an immune response in solid tumors after local injection of the adenoviral/p53 gene which elicits an immune reaction leading to tumor necrosis (Ko, et al., 1996).
Protein p53 appears to be a transcription factor able to recognize specific regulatory regions in a number of genes via its central DNA-binding domain; the DNA sequence-specific binding of wt p53 is regulated by the C-terminal domain of p53 and is activated by a variety of posttranslational modifications (Hupp, et al., 1992; reviewed by Hupp and Lane, 1994).
Increased levels of p53 up regulate the expression of specific genes including Cip-1/Waf-1/p21 (El-Deiry, et al., 1993), GADD45 (Kastan, et al., 1992), cyclin G (Okamoto and Beach, 1994), and mdm2 (Perry, et al., 1993; Barak, et al., 1993; Momand, et al., 1992) which is induced by UV damage in a p53-dependent pathway (Perry, et al., 1993). Other genes up-regulated by p53 include human PCNA (Shivakumar, et al., 1995), mouse muscle creatine kinase MCK (Zambetti, et al., 1992), EGFR (Deb, et al., 1994), GADD45 (Kastan, et al., 1992), the potent promoter of the death pathway Bax (Miyashita and Reed, 1995), and thrombospondin-1 (Dameron, et al., 1994).
Mdm2 acts as a feedback loop for the biological functions of p53 apparently to moderate the G1/S arrest or apoptosis triggered by p53 following severe damage to DNA. Mdm2 protein associates with p53 causing p53 inactivation by preventing its sequence-specific binding to regulatory targets in DNA (Momand, et al., 1992; Oliner, et al., 1992). Elevated levels of Mdm2 mimic the effect of T antigen, E1l B of adenovirus, E6 of HPV, which also inactivate p53 in a similar manner; overexpression of Mdm2 can block the induction of apoptosis by p53 (Chen, et al., 1994).
Gadd45 is believed to inhibit cell cycle progression; however, the mechanism has not been elucidated (Papathanasiou, et al., 1991).
The PCNA promoter is up-regulated in the presence of moderate amounts of wt p53; however, at higher levels of wt p53 the PCNA promoter is inhibited whereas tumor-derived p53 mutants activate the PCNA promoter (Shivakumar, et al., 1995); it has been suggested that the moderate elevation in wt p53 seen after DNA damage induces PCNA to cope with its DNA repair activities (Shivakumar, et al., 1995); this inhibition in DNA replication but stimulation in repair by p53 might be accomplished by an independent pathway involving induction of p21 (El-Deiry, et al., 1993) which interacts with PCNA protein auxiliary to DNA polymerase d to inhibit the replication but not the repair functions of PCNA (Li, et al., 1994).
The bax gene which induces apoptosis is upregulated by p53 whereas the bcl-2 gene which inhibits apoptosis in B cells is down-regulated by p53 (Miyashita, et al., 1994a, 1994b; Miyashita and Reed, 1995). Initiated cancer cells may lead to tumor development only when a dysfunction in their apoptotic pathway takes place; some of the mechanisms leading to inactivation of the apoptotic pathway in cancer cells may result from an up-regulation in the bcl-2 gene (a Bcl-2 chimeric factor is produced in leukemias as a result of a translocation) or down-regulation of the bax gene. Gene therapy for cancer could involve restoration of the apoptotic pathway in cancer cells leading to their suicidal death; this could be effected by overexpression of the bax gene or in the suppression of the endogenous bcl-2 gene for example using p53 expression vectors).
p53 binding sites have been found at the origin of replication of polyomavirus with an inhibitory effect on virus replication in vitro (Miller, et al., 1995) and at the SV40 ORI (Bargonetti, et al., 1991) as well as in putative cellular origins of replication (Kern, et al., 1991). p53 interacts with replication protein A (RPA) (implicated in DNA replication and in repair; interaction of p53 inhibits the replication functions of RPA (Dutta, et al., 1993) although interaction of p53 with RPA via its acidic domains stimulate BPV-1 DNA replication in vitro (Li and Botchan, 1993). Immunolocalization of p53 (also of RB and host replication proteins) at foci of viral replication in HSV-infected cells (Wilcock and Lane, 1991) provided further evidence for a direct interaction of p53 with proteins (or DNA sequences) at the replication fork. Wild-type p53 suppressed DNA replication in vitro when the p53 binding site (RGC).sub.16 from the ribosomal gene cluster was cloned on the late side of the polyomavirus (Py) core origin; when mutated p53-binding sites were used, the inhibition in Py replication was not observed. In addition, RPA (able to interact directly with p53) was unable to relieve the p53-mediated repression in Py replication.
The tumor suppressor p53 has the ability to recognize via its C-terminal domain DNA insertion/deletion mismatches consisting of one or a few extra bases on one strand (Lee, et al., 1995).
p53 binds to strand breaks in DNA (Lu and Lane, 1993; Nelson and Kastan, 1994); electron microscopy studies have shown that the C-terminal domain of p53 binds directly to ends of single-stranded DNA whereas the central domain of p53 binds to more internal segments (Bakalkin, et al., 1995). Short single strands considerably stimulate the sequence-specific binding of p53 to its cognate sites in supercoiled DNA and this recognition also involves the C-terminal domain of p53 (Jayaraman and Prives, 1995); a 29 nt segment of DNA known to arise by two endonuclease cuts during NER in mammalian cells around the lesion could stimulate p53 binding to DNA and might play some physiological function in the subsequent steps of repair (Jayaraman and Prives, 1995).
II. Differences in Biological Functions Between Wild-Type p53 and Tumor-Derived p53 Mutants
Tumor-derived mutant forms of p53 have lost their DNA sequence-specific binding capacities. For example the Trp-248 and His-273 mutants of p53 have poor DNA-binding abilities and are unable to activate transcription from constructs containing p53 binding sites (Farmer, et al., 1992).
A. Wild Type
Wild-type (wt) p53 tumor suppressor protein negatively regulates cell growth (Hollstein, et al., 1991; Prives, 1994). Whereas the wild-type p53 acts as a tumor suppressor, several of the mutant forms display oncogenic activities (Levine, 1993; Prives and Manfredi, 1993; Deppert, 1994). Although the wt p53 has been postulated to repress growth by activating genes that repress growth (p21), many of the mutant forms have lost their DNA sequence-specific binding and transcriptional activation capacities (reviewed by Zambetti and Levine, 1993).
According to one model (see Vogelstein and Kinzler, 1992), wt p53 is a positive regulator for the transcription of genes that by themselves are negative regulators of growth control and/or invasion. Indeed, p53 upregulates the genes of p21/CIP1/WAF1(El-Deiry, et al., 1993) and GADD45 (Kastan, et al., 1992) whose products interact with PCNA to inhibit its association with DNA polymerase d thus causing arrest in DNA replication (Waga, et al., 1994; Smith, et al., 1994). This feature of p53 that is central to its ability to suppress neoplastic growth is lost by mutations on p53 that result in loss of its ability to bind to DNA or to interact with other transcription protein factors (see also Farmer, et al., 1992; Kern, et al., 1992).
B. Mutant p53
Mutant p53 can transactivate genes that up-regulate cellular growth (Deb, et al., 1992; Dittmer, et al., 1993) such as PCNA (Shivakumar, et al., 1995), EGFR (Deb, et al., 1994), multiple drug resistance (MDR1) (Chin, et al., 1992; Zastawny, et al., 1993), and human HSP70 in vivo (Tsutsumi-Ishi, et al., 1995). These studies support the idea for an oncogene function of the mutant p53 protein compared with the tumor suppressor function of wt p53; mutation in the p53 gene may, thus, cause gain of new functions such as transforming activation and binding to a distinct class of promoters which are not normally regulated by wt p53 (Zambetti and Levine, 1993; Tsutsumi-Ishi, et al., 1995). At the same time appearance of mutations in the p53 gene result in the loss of function of the wt p53 (Zambetti and Levine, 1993).
The wild-type but not mutant p53 at low levels transactivates the human PCNA promoter in a number of different cell lines; the wild-type p53-response element from the PCNA promoter functions in either orientation when placed on a heterologous synthetic promoter; thus moderate elevation of p53 can induce PCNA, enhancing the nucleotide excision repair functions of PCNA (Shivakumar, et al., 1995). Whereas low levels of wild-type p53 activate the PCNA promoter, higher concentrations of wt p53 inhibit the PCNA promoter, and tumor-derived p53 mutants activate the promoter (Shivakumar, et al., 1995).
SV40 T antigen was unable to act as an initiator of SV40 DNA replication in vitro when complexed with p53 (Wang, et al., 1989); mutant p53 was unable to cause inhibition in the initiating functions of T antigen in vitro (Friedman, et al., 1990).
While the wt p53 is endowed with a 3'-to-5' exonuclease activity, associated with the central DNA-binding domain, and thought to function during repair, replication, and recombination; the 273.sup.His mutant of p53 has lost the exonuclease activity (Mummenbrauer, et al., 1996).
C. Regulation of the p53 Gene
Very few studies on the regulation of the p53 gene are today available (Deffie, et al., 1993; Stuart, et al., 1995). The p53 gene is activated by the wt p53 but not by the functionally inactive mutant p53 protein; mobility shift assays and methylation interference have pinpointed the +22 to +67 region of the promoter of the p53 gene responsible for up-regulation containing an NF-.kappa.B response element and a p53-binding site at 10 of 11 nucleotides (Deffie, et al., 1993); it has been difficult to demonstrate direct binding of p53 to this regulatory region of its own gene and thus p53 may transactivate one or more transcription factors such as PRDII-BF1 (also known as MBP1 and HIVEP1), .alpha.A-CRYBP1, and AGIE-BP1 as well as NF-.kappa.B that bind the NF-.kappa.B response element (Deffie, et al., 1993).
p53 is down-regulated by Pax5 in early steps during embryogenesis is by interacting with a DNA control element within exon 1 of the p53 gene; at later stages of embryogenesis and in bone marrow cells Pax5 expression drops allowing the levels of p53 to rise; increased p53 was proposed to induce either apoptosis or B cell differentiation to plasma cells (Stuart, et al., 1995).
III. Gene Therapy Strategies Based on p53
A. Transfer of the p53 Tumor Suppressor Gene to Cancer Cells
Preclinical studies have shown that both viral and plasmid vectors able to mediate high efficiency delivery and expression of wild-type tumor suppressor p53 gene can cause regression in established human tumors, prevent the growth of human cancer cells in culture, or render malignant cells from human biopsies non-tumorigenic in nude mice. Inhibition in cell proliferation was observed in cell culture and in tumors after induction of p53 expression with adenovirus vectors (Bacchetti and Graham, 1993; Wills, et al., 1994). Intratracheal injection of a recombinant retrovirus containing the wt p53 gene was shown to inhibit the growth of lung tumors in mice nu/nu models inoculated intratracheally with human lung cancer H226Br cells whose p53 gene has a homozygous mutation at codon 254 (Fujiwara, et al., 1994). A number of other studies have shown suppression in tumor cell growth and metastasis after delivery and expression of the wt p53 gene (Diller, et al., 1990; Chen, et al., 1991; Isaacs, et al., 1991). A human clinical trial at M. D. Anderson Cancer Center uses transfer of the wild-type p53 gene in patients suffering with non-small cell lung cancer and shown to have p53 mutations in their tumors using local injection of an Ad5/CMV/p53 recombinant adenovirus at the site of tumor in combination with cisplatin (Roth, et al., 1996).
Delivery of the p53 gene to malignant human breast cancer cells in nude mice using DOTMA:DOPE 1:1 cationic liposomes (400 nmoles liposomes/35 mg DNA) resulted in regression (60% reduction in tumor cell volume) in 8 out of 15 animals treated; animals were receiving one injection every 10 days (Lesoon-Wood, et al., 1995). It was thought that wild-type p53 expression (tumor cells were expressing mutant forms of p53) upregulated p21 gene to inhibit cell growth by inhibition in cyclin-dependent kinases but also via induction of apoptosis preferentially in cancer cells.
B. Delivery of p53 to Prostate Cancer Cells
Prostate cancer cells have a mutated p53 gene: three of five prostate cancer cell lines examined (TSUPr-1, PC3, DU145) and one out of two primary prostate cancer specimens were found to harbor mutations altering the amino acid sequence of the conserved exons 5-8 of the p53 gene; transduction of the p53-defective cell lines with the wt p53 gene using lipofectin showed reduction in tumorigenicity assayed from reduced colony formation and the cells became growth arrested (Isaacs, et al., 1991). Although primary prostate tumors have few mutations in the p53 gene (Voeller, et al., 1994; Isaacs, et al., 1994), specimens from advanced stages of the disease and metastases as well as their cell lines frequently display mutations or deletions at both alleles of the p53 gene (Chi, et al., 1994; Dinjens, et al., 1994).
Introduction of the wt p53 or of the p21 downstream mediator of p53-induced growth suppression into a mouse prostate cancer cell line, deficient for p53, led to an association of p21 with Cdk 2; this interaction was sufficient to downregulate Cdk 2 by 65% (Eastham, et al., 1995). The p21 gene, driven by CMV promoter into an Adenovirus 5 vector, was more effective than the AD5CMV-p53 vector, under control of the same elements as p21, in reducing tumor volume in syngeneic male mice with established s.c. prostate tumors. Tumors were induced by injection of 2 million cells in each animal. These studies suggest that p21 expression might have more potent growth suppressive effect than p53.
Infection of the androgen-independent human prostate Tsu-pr1 cell line lacking functional p53 alleles with recombinant adenovirus vectors (replication-deficient) carrying the p53 gene under control of the CMV promoter resulted in expression of p53 and induced striking morphological changes: the cells were detached from the substratum, condensed, and exhibited breakdown of the nuclear DNA into nucleosome-size fragments characteristic of apoptosis; whereas control cells were able to elicit tumors in nude mice, the AdCMV/p53-infected cells failed to form tumors (Yang, et al., 1995).
Endocrine therapy is ineffective once the prostate cancer becomes androgen-independent; these cancers remain unresponsive to conventional chemotherapy. Androgen-independent and metastatic prostate cancers were established in athymic male mice by co-inoculation with the LNCaP human prostate cancer cell line and the MS human bone stromal cell line; these tumors became necrotic and were successfully eradicated by intratum oral injection of a recombinant p53/adenovirus; the p53 gene was driven by the CMV promoter and the SV40 poly(A) signal placed in the E1 region of Ad5 (Ko, et al., 1996). It was suggested that in addition to the tumor suppressor, apoptotic, and antiangiogenesis function of p53, tumor necrosis was induced by a bystander effect or a general immune response which attracted immune cells to cause tumor cell killing (Ko, et al., 1996).
However, a significant factor to be considered in these approaches is the competition of the wt p53 functions by the endogenous mu p53 expressed in tumor cells; optimal results will be expected if the endogenous mu p53 gene is inhibited with the simultaneous overexpression of the wt p53 gene.
As mutated forms of the p53 tumor suppressor gene are the most frequent in human tumors, down-regulation of the mutated endogenous p53 gene in prostate cancer cells and the simultaneous transfer and expression of a wild-type p53 gene able to undertake the tumor suppressor functions is expected to be of therapeutic value inducing apoptotic death to the prostate cancer cells.
C. Cancer Treatment by Transduction of Suicidal HSV-tk Gene Using Liposomes Followed by Treatment with Ganciclovir
The cells in a solid tumor are of different pheno- and geno-types; such differences among cells in the same tumor may include differences in ploidy, rearrangements, translocations, expression of oncogenes or tumor suppressor genes, and spectra of mutations among the various genes. It might therefore be an advantage simply to develop strategies for killing tumor cells rather than correct defective genes that led to the cancer phenotype; a package of additional mutations and/or changes may have accumulated in these cells.
A rather successful gene transfer approach results in the direct suppression of tumor growth by cytotoxic gene therapy. Cancer cells can be induced to be conditionally sensitive to the antiviral drug ganciclovir after their transduction with the thymidine kinase (tk) gene from the herpes simplex virus (HSV); ganciclovir is the 9-{[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyl}guanine (Field, et al., 1983); it is converted by HSV-tk into its monophosphate form which is then converted into its triphosphate form by cellular enzymes and is then incorporated into the DNA of replicating mammalian cells leading to inhibition in DNA replication and cell death (Moolten, 1986; Borrelli, et al., 1988; Moolten and Wells, 1990).
It is only viral TK, not the mammalian enzyme, that can use efficiently ganciclovir as a substrate and this drug has been synthesized to selectively inhibit herpes virus replication (Field, et al., 1983); indeed, the mammalian TK has a very low affinity for this guanosine analog. The toxicity of ganciclovir is manifested only when cells undergo DNA replication and it is not harmful to normal nondividing cells. This treatment strategy has been used for hepatocellular carcinoma (Huber, et al., 1991; Su, et al., 1996), fibrosarcoma, glioma (Culver, et al., 1992, see below), adenocarcinoma (Osaki, et al., 1994) and prostate cancer (Eastham, et al., 1996).
This patent proposes other suicidal genes instead of HSV-tk such as the cytosine deaminase (CD) gene. The CD protein catalyzes the conversion of the prodrug 5-fluorocytosine (5FC) to 5-fluorouracil (5FU); treatment of cells, transfected with this construct, with 5FC results in the conversion of the 5FC into the antitumor drug 5FU into CD-positive tumor cells (Mullen, et al., 1992; Austin and Huber, 1993; Huber, et al., 1993; 1994; Richards, et al., 1995).
This approach has been used for the treatment of primary and metastatic hepatic tumors based on the overexpression of the suicidal CD gene under control of the regulatory regions of the tumor marker gene carcinoembryonic antigen (Richards, et al., 1995, see below).